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Proteins and kits for tuberculosis diagnosis

A protein and nucleotide technology, applied in the fields of biochemistry and immunology, can solve the problems that cannot be used as a diagnostic basis, and are prone to false positive rates and high false positive rates.

Inactive Publication Date: 2016-12-28
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is easy to be confused with other lung diseases and needs to be confirmed by a professional doctor
(3) Immunological diagnosis of tuberculosis: 1. The tuberculin pure protein derivative (PPD) test is commonly used. A positive test is one of the evidences of tuberculosis infection, but the false positive rate is high and it is easy to misdiagnose
2. Positive tuberculosis antibody tests in blood and sputum are also helpful for diagnosis, and false positive rates are also prone to occur
5. Polymerase chain reaction (PCR), the advantage is that the sensitivity can reach 98%-100%, and the disadvantage is that the specificity is poor
(4) Other examinations: can only be used as an auxiliary diagnosis, not as a basis for diagnosis

Method used

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  • Proteins and kits for tuberculosis diagnosis
  • Proteins and kits for tuberculosis diagnosis
  • Proteins and kits for tuberculosis diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Cloning and expression of related genes of MTB H37Rv (Gateway method)

[0064] Invitrogen's Gateway technology provides a possibility for protein recombination and expression. It starts from PCR products to create Gateway TM Getting started with cloning. This method is achieved by incorporating attB sites into the upstream and downstream primers, and then co-incubating the PCR amplified product with pDONR TM (including attP site) and Gateway TM BP Clonase TM Enzyme mix. Then transformed into Escherichia coli, the entry clone containing the target gene will be obtained, and attL recombination sites are flanked on both sides of the target gene. This starter clone can be used with Gateway TM The target vector pDEST17 is recombinantly expressed to obtain the target protein quickly and efficiently [Walhout AJ, Temple GF, Brasch MA. et al. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods ...

Embodiment 2

[0174] Example 2: Protein expression and purification

[0175] 1. Experimental method

[0176] Protein purification is a necessary condition for downstream experiments. Prokaryotic cells can contain thousands of different proteins after expression, some are very abundant, and some contain only a few copies. In order to study a certain protein, the protein must first be purified from other proteins and non-protein molecules in order to remove false positives caused by foreign foreign proteins. High-efficiency and high-purity proteins require high-throughput protein purification. The expressed protein in this experiment is a prokaryotic expression containing a 6His tag, so the affinity of histidine adjacent to the His.Tag sequence and the immobilized metal nickel ion is used for purification.

[0177] 1. Resurrection and amplification of strains

[0178] Prepare the LB medium required for strain recovery and expansion, and add corresponding antibiotics after it cools down. Th...

Embodiment 3

[0201] Example 3: ELISPOT analysis

[0202] Enzyme-linked immunospot (ELISPOT) technology is the best technology for detecting the level of cellular immunity in the world today. It integrates many advantages such as high sensitivity, high reliability, high throughput, single cell level, functional detection and low cost. It has been widely used in immunology circles at home and abroad, and has become one of the mainstream immunological detection technologies. The principle of ELISPOT detection is as follows:

[0203] A. Coat the bottom of the detection well with anti-γ-interferon monoclonal antibody.

[0204] B. Isolating lymphocytes from the sample to be tested.

[0205] C. Put the cells to be tested into the detection wells and culture them for about 20 hours, and add the antigen (stimulator) at the same time. During the culture period, T lymphocytes that specifically react to the antigen will be activated and begin to secrete specific cytokines (such as γ-interferon), wh...

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Abstract

The invention belongs to the fields of biochemistry and immunology, relates to a protein and a kit for tuberculosis diagnosis, and particularly relates to a separated protein shown as the following amino acid sequence SEQ ID NO:1 or SEQ ID NO:2. The protein can be used as a molecular marker for cell-level tuberculosis immunodiagnosis, and has a good accordance rate and intensity of reaction.

Description

technical field [0001] The invention belongs to the fields of biochemistry and immunology, and relates to a protein and a kit for diagnosing tuberculosis. Background technique [0002] Mycobacterium tuberculosis is one of the most threatening pathogens to human health. Tuberculosis is the main infectious disease among adults in the world today and has posed a serious challenge to international public health. After infecting the human body, Mycobacterium tuberculosis is mostly in a latent state or a persistent infection state. It can evade the host's defense in the infected host cells and reproduce in large numbers and achieve a delicate balance with the host. May be ill. Under normal circumstances, 10% of infected people will develop active tuberculosis. An untreated active tuberculosis patient can infect 10-15 people a year. [0003] In the past 50 years, the theory and technology of tuberculosis control, clinical diagnosis and treatment level and research have made grea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/35C07K16/12C12N15/31C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12N15/11A61K39/40A61K39/04A61K48/00A61P31/06C12Q1/68C12Q1/04G01N33/68G01N33/569
CPCA61K39/00A61K2039/505C07K14/35C07K16/1289C12Q1/689G01N33/5695G01N33/6893G01N2500/04
Inventor 金奇刘立国张维佳张笑冰
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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